Evaluation of a Novel Immunochromatographic Assay Using Monoclonal Antibodies against the Matrix Protein of Human Metapneumovirus.

BACKGROUND: The aim of this study was to determine the sensitivity and specificity of a novel immunochromatographic (IC) assay (APD1806) using monoclonal antibodies against the matrix (M) protein of human metapneumovirus (hMPV) for detection of hMPV from nasopharyngeal swab samples based on the results of real-time RT-PCR. METHODS: Nasopharyngeal swab samples taken from 189 patients aged 0 - 5 years who were suspected of having respiratory tract infections associated with hMPV were used in this study. The samples were tested both by the IC assay and by real-time RT-PCR for detection of hMPV. RESULTS: The sensitivity and specificity of the IC assay for detection of hMPV were 88.8% (95/107) and 92.7% (76/82), respectively. CONCLUSIONS: The IC assay using monoclonal antibodies against the M protein of hMPV is an accurate and fast assay that is suitable as a diagnostic tool for hMPV infection. The optimal timing of the IC assay is 12 hours or more after the onset of fever due to hMPV infection.

P1 gene of Mycoplasma pneumoniae isolated from 2016 to 2019 and relationship between genotyping and macrolide resistance in Hokkaido, Japan.

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019.

Unusual mono-reassortant of a Wa-like G1P[8] species A rotavirus containing a DS-1-like (genotype 2) NSP4 gene.

Species A rotaviruses are a major cause of acute gastroenteritis in infants and young children worldwide. Reassortment is a common phenomenon due to the segmented nature of the rotavirus genome. The complete coding sequences of a species A rotavirus strain isolated from the feces of a child with acute gastroenteritis in Japan in 2018 were determined using an unbiased viral metagenomics approach. The genetic analysis revealed that the rotavirus strain had an unusual genomic constellation (G1-P[8]-I1-R1-C1-M1-A1-N1-T1-E2-H1), suggesting reassortment of a genotype 1 with a genotype 2 rotavirus, from which the NSP4-encoding gene was acquired.

Distribution of rotavirus genotypes in Japan from 2015 to 2018: Diversity in genotypes before and after introduction of rotavirus vaccines.

BACKGROUND: Diversity in group A rotavirus (RVA) strains after introduction of RV-vaccines remains an emerging concern worldwide. In this study, we investigated the prevalence and distribution of RVA genotypes in Japanese children with acute gastroenteritis (AGE) from 2015 to 2018. In addition, a comparison of the genotypes in pre-vaccination (2006-2012) and post-vaccination (2012-2018) periods was conducted to understand the impact of these vaccines on genotype distribution. METHODS: Fecal samples were collected regularly from outpatient clinics in six localities: Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga. RVA were screened and genotyped by RT-PCR and sequence-based genotyping. RESULTS: During the period 2015-2018, RVA was detected in 307 (19.7%) samples out of 1557 specimens: 29.9% (95% CI: 25.8% to 34.3%), 17.9% (95% CI: 14.7% to 21.5%), and 13% (95% CI: 10.3% to 16.0%) were detected RVA-positive in 2015-2016, 2016-2017 and 2017-2018, respectively. The average detection of RVA in pre-vaccination (2006-2012) and post-vaccination (2012-2018) era remained almost similar (18%-20%). The G2P[4]I2 (52.1%, 95% CI: 43.5%-60.6%) remained the most common genotype in 2015-2016, whereas G8P[8]I2 (55.9%, 95% CI: 45.2%-66.2%) dominated in 2016-2017. In 2017-2018, G9P[8]I2 (42.0%, 95% CI: 30.5%-53.9%) prevailed, followed by G9P[8]I1 (23.0%, 95% CI: 14.0%-34.2%). The detection rate of some common genotypes of pre-vaccination era like G1P[8] and G3P[8] has been reduced after introduction of RV-vaccine, whereas genotypes that were sporadic before the introduction of vaccines like G2P[4], G2P[8], G9P[8] and G8P[8] were emerged/reemerged in post-vaccination period. CONCLUSIONS: Our study presented the diversity in circulating RVA genotypes in Japan before and after introduction of RV-vaccines. Sudden emergence of DS-1-like (I2) unusual strains in post-vaccination era remains alarming. Continuous monitoring of RVA genotypes is therefore indispensable to refine future vaccine strategy.

Consensus and variations in cell line specificity among human metapneumovirus strains.

Human metapneumovirus (HMPV) has been a notable etiological agent of acute respiratory infection in humans, but it was not discovered until 2001, because HMPV replicates only in a limited number of cell lines and the cytopathic effect (CPE) is often mild. To promote the study of HMPV, several groups have generated green fluorescent protein (GFP)-expressing recombinant HMPV strains (HMPVGFP). However, the growing evidence has complicated the understanding of cell line specificity of HMPV, because it seems to vary notably among HMPV strains. In addition, unique A2b clade HMPV strains with a 180-nucleotide duplication in the G gene (HMPV A2b180nt-dup strains) have recently been detected. In this study, we re-evaluated and compared the cell line specificity of clinical isolates of HMPV strains, including the novel HMPV A2b180nt-dup strains, and six recombinant HMPVGFP strains, including the newly generated recombinant HMPV A2b180nt-dup strain, MG0256-EGFP. Our data demonstrate that VeroE6 and LLC-MK2 cells generally showed the highest infectivity with any clinical isolates and recombinant HMPVGFP strains. Other human-derived cell lines (BEAS-2B, A549, HEK293, MNT-1, and HeLa cells) showed certain levels of infectivity with HMPV, but these were significantly lower than those of VeroE6 and LLC-MK2 cells. Also, the infectivity in these suboptimal cell lines varied greatly among HMPV strains. The variations were not directly related to HMPV genotypes, cell lines used for isolation and propagation, specific genome mutations, or nucleotide duplications in the G gene. Thus, these variations in suboptimal cell lines are likely intrinsic to particular HMPV strains.

Evaluation of a Novel Immunochromatographic Assay for Detection of Human Adenoviruses from Throat Swab Samples Compared with Real-time PCR.

BACKGROUND: The aim of this study was to determine the sensitivity and specificity of a novel immunochromatographic assay (ICA) kit, ALSONIC® Adeno (Alfresa Pharma Co., Osaka, Japan), for the detection of human adenovirus (HAdV) from throat swab samples based on the results of real-time PCR. The incubation time required for the novel assay kit (5 minutes) is shorter than that required for other ICA kits that are available in Japan. METHODS: Throat swab samples were taken from 151 patients aged 6 months to 15 years who were suspected of having respiratory tract infections caused by HAdV. RESULTS: The sensitivity and specificity of the ICA for detection of HAdV were 92.2% (83/90) and 95.1% (58/61), respectively, and the assay showed positive and negative predictive values of 96.5% (83/86) and 89.2% (58/65), respectively. CONCLUSIONS: ALSONIC® Adeno is suitable as a diagnostic tool in the acute phase of HAdV infection.

Clinical effectiveness of four neuraminidase inhibitors (oseltamivir, zanamivir, laninamivir, and peramivir) for children with influenza A and B in the 2014-2015 to 2016-2017 influenza seasons in Japan.

The clinical effectiveness of four neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, laninamivir, and peramivir) for children aged 0 months to 18 years with influenza A and B were investigated in the 2014-2015 to 2016-2017 influenza seasons in Japan. A total of 1207 patients (747 with influenza A and 460 with influenza B) were enrolled. The Cox proportional-hazards model using all of the patients showed that the duration of fever after administration of the first dose of the NAI was shorter in older patients (hazard ratio = 1.06 per 1 year of age, p < 0.001) and that the duration of fever after administration of the first dose of the NAI was shorter in patients with influenza A infection than in patients with influenza B infection (hazard ratio = 2.21, p < 0.001). A logistic regression model showed that the number of biphasic fever episodes was 2.99-times greater for influenza B-infected patients than for influenza A-infected patients (p < 0.001). The number of biphasic fever episodes in influenza A- or B-infected patients aged 0-4 years was 2.89-times greater than that in patients aged 10-18 years (p = 0.010), and the number of episodes in influenza A- or B-infected patients aged 5-9 years was 2.13-times greater than that in patients aged 10-18 years (p = 0.012).

Molecular epidemiology of rotavirus gastroenteritis in Japan during 2014-2015: Characterization of re-emerging G2P[4] after rotavirus vaccine introduction.

Rotavirus vaccines have been available in Japan since 2011. This study conducted to monitor the trend of group A rotavirus (RVA) genotypes 3 years after vaccine introduction. A total of, 539 fecal samples were collected from children with acute gastroenteritis in six regions during July 2014-June 2015. Among them, 178 samples (33.0%) were positive for RVA. The most predominant genotype was G1P[8] (35.9%) followed by G2P[4] (26.4%), G9P[8] (21.3%), G3P[8] (4.5%), and G3P[9] (4.5%). The detection rate of G2P[4] was increased soon after vaccine introduction. Sequence analyses of VP7 and VP4 genes of the representative G2P[4] strains were found to be clustered in sub-lineage IVa of lineage IV. It is noteworthy that one amino acid substitution in the antigenic epitope (Q114P) of VP4 gene was found in representative G2P[4] strains of the current study. However, it is unclear whether the change in antigenic epitope is due to the effect of vaccination or due to natural variation, warranting further continuous monitoring of rotavirus evolution after vaccine introduction.

Molecular Epidemiological Traits of Group A Rotaviruses in Japanese Children During Transitional Period of Rotavirus Vaccine Implementation, 2011 - 2014.

BACKGROUND: Group A rotavirus (RVA) vaccines have been introduced in Japan since 2011. To investigate the molecular epidemiological traits of RVA during the transitional period of rotavirus vaccine implementation in Japan, this study was undertaken by following up three-decade long surveillance conducted in the same regions. METHODS: RVA were screened and genotyped by RT-PCR from diarrheal samples collected from non-hospitalized patients in six localities (Hokkaido, Tokyo, Shizuoka, Osaka, Kyoto, and Saga) during 2011 - 2014. Selected samples were sequenced to elucidate the evolutionary trend. RESULTS: Among 1858 specimens, the detection rate of RVA declined to 4.0% in 2013 - 2014 from 17.9% in 2011 - 2012 and 22.1% in 2012 - 2013. G1P[8] was the most predominant genotype in the first two years accounting for more than half, and G9P[8] showed the highest detection rate as 35.0% in the last year. Interestingly, the proportional rate of G2 strains in the studied period increased from 0% to 25%. VP6 genotyping revealed that DS-1 like reassortant G1P[8] strains were detected all over Japan and their prevalence fluctuated greatly from 35.0% to 89.5%. Sequence analysis of VP6 showed that strains in the current strains were closely related but distinct from the original reference strains, namely Wa and DS-1. CONCLUSIONS: The detection rates of RVA, their GP combinations, prevalence of reassortant strains varied greatly after the introduction of rotavirus vaccines in Japan. Continuous monitoring is warranted to refine future vaccine strategy.

Therapeutic efficacy of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant and macrolide-sensitive Mycoplasma pneumoniae pneumonia in pediatric patients.

OBJECTIVE: To clarify therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin against macrolide-resistant Mycoplasma pneumoniae (MRMP) pneumonia and against macrolide-sensitive Mycoplasma pneumoniae (MSMP) pneumonia in pediatric patients. METHODS: A prospective, multicenter observational study was conducted from July 2013 to August 2015. The therapeutic effects of azithromycin, clarithromycin, minocycline and tosufloxacin were evaluated in 59 patients with pneumonia caused by MRMP and in 50 patients with pneumonia caused by MSMP. In vitro activities of antimicrobial agents against isolates of Mycoplasma pneumoniae were also measured. RESULTS: Mean durations of fever following commencement of treatment in patients infected with MRMP and MSMP were 5.2 and 1.9 days, respectively (log-rank test, P < 0.0001). Among patients infected with MRMP, mean durations of fever were 4.6, 5.5, 1.0 and 7.5 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P < 0.0001). Among patients infected with MSMP, mean durations of fever were 2.5, 1.7, 0.9 and 4.3 days for patients treated with azithromycin, clarithromycin, minocycline and tosufloxacin, respectively (log-rank test, P = 0.0162). The MIC90s of azithromycin and clarithromycin among the 27 isolates of MRMP were 64 and 256 µg/ml, respectively, and those among the 23 isolates of MSMP were <0.000125 and 0.001 µg/ml, respectively. The MIC90s of minocycline and tosufloxacin among the 27 isolates of MRMP were 1.0 and 0.25 µg/ml, respectively, and those among the 23 isolates of MSMP were 1.0 and 0.5 µg/ml, respectively. CONCLUSION: Both minocycline and tosufloxacin showed good in vitro activities against MRMP. Minocycline, but not tosufloxacin, shortened the duration of fever in pediatric patients infected with MRMP compared to the duration of fever in patients treated with macrolides.

Regional Differences in Prevalence of Macrolide Resistance among Pediatric Mycoplasma pneumoniae Infections in Hokkaido, Japan.

Recently, macrolide-resistant (MR) Mycoplasma pneumonia appeared, and prevalence of macrolide resistance among M. pneumoniae infections varies by country. However, reports on regional differences in the prevalence of MR M. pneumonia within a country are scarce. In this study, 617 nasopharyngeal swab samples were collected from 617 pediatric patients, and DNA of M. pneumoniae was identified in 95 samples. In 51 of the 95 M. pneumonia positive samples, we detected the presence of mutation A2063G mutation (conferring macrolide resistance) in the 23S rRNA gene. The overall macrolide resistance rate was 53.7%, but there were regional differences: 0.0% in Muroran, 5.3% in Asahikawa, 55.3% in Sapporo, and 100.0% in Kushiro. Statistically significant pairwise differences in the prevalence of MR M. pneumoniae were observed among these cities except for the pair of Muroran and Asahikawa. After exclusion (in order to avoid the influence of macrolides) of patients who were prescribed macrolides before collection of nasopharyngeal swab samples, statistically significant differences persisted: 0.0% in Muroran, 5.6% in Asahikawa, 38.5% in Sapporo, and 100.0% in Kushiro.

Sensitivity and Specificity of a Loop-Mediated Isothermal Amplification Assay for the Detection of Mycoplasma Pneumonia from Nasopharyngeal Swab Samples Compared with those of Real-time PCR.

BACKGROUND: The aim of this study was to determine the sensitivity and specificity of a loop-mediated isothermal amplification (LAMP) assay kit for the detection of Mycoplasma pneumonia (Eiken Chemical Co., Ltd, Tokyo, Japan) from nasopharyngeal swab samples compared with those of real-time PCR. METHODS: Nasopharyngeal swab samples taken from 223 patients aged 3 - 18 years who were suspected of having respiratory tract infections associated with Mycoplasma pneumonia were used in this study. The samples were tested both by the LAMP assay and by real-time PCR for detection of Mycoplasma pneumonia. RESULTS: The sensitivity and specificity of the LAMP assay for the detection of Mycoplasma pneumonia were 99.1% (105/106) and 100.0% (117/117), respectively. CONCLUSIONS: The LAMP assay for the detection of Mycoplasma pneumonia is an accurate and fast assay that is suitable as a diagnostic tool in the acute phase of Mycoplasma pneumonia infection.

Comparison of the clinical effectiveness of zanamivir and laninamivir octanoate for children with influenza A(H3N2) and B in the 2011-2012 season.

OBJECTIVES: To evaluate the clinical effectiveness of the two inhaled neuraminidase inhibitors (NAIs), zanamivir (ZN) and laninamivir octate (LO), for influenza A(H3N2) and B virus infections. DESIGN: A prospective, multicenter observational study was conducted from January to April in 2012. SETTING: Outpatients aged 5-18 years who had a temperature of 37.5°C or higher and were diagnosed as having influenza based on an immunochromatographic assay were enrolled. SAMPLE: A total of 338 patients treated with ZN and 314 patients treated with LO were compared. MAIN OUTCOME MEASURES: The duration of fever after administration of the first dose of each NAI was evaluated as a primary endpoint. The secondary endpoint was episodes of biphasic fever. RESULTS: No statistically significant difference in the duration of fever was found between the ZN and LO groups (log-rank test, P = 0.117). A logistic regression model showed that episodes of biphasic fever increased by 1.19 times for every decrease of 1 year of age (P = 0.016) and that the number of biphasic fever episodes in patients treated with LO was 5.80-times greater than that in patients treated with ZN (P < 0.001). CONCLUSIONS: Although the duration of fever in the LO group was comparable to that in the ZN group, episodes of biphasic fever were more frequent in younger children and in the LO group than in the ZN group.

Reemergence of new variant G3 rotavirus in Japanese pediatric patients, 2009-2011.

The molecular epidemiology and characterization of rotaviruses obtained from non-hospitalized children with acute gastroenteritis in five different prefectures (Hokkaido, Saga, Tokyo, Osaka, and Kyoto) from July 2009 to June 2011 was investigated. Among 831 fecal specimens tested, rotavirus was found in 165 specimens (19.9%). The rotavirus detection rate in 2010-2011 (23.3%) was higher than those in 2009-2010 (16.0%). The highest prevalence of rotavirus was found in children aged 12 to 23 months. Rotavirus could be detected throughout the 8 month period with a peak in April. We found that G3P[8] was the most prevalent genotype (54.5%), followed by G1P[8] (29.1%), G9P[8] (9.1%), G3P[4] (3.0%), G2P[4] (2.5%), G1P[4] (1.2%), and G4P[8] (0.6%), respectively. Interestingly, G3 strains emerged as the most predominant genotype and replaced G1 rotavirus which had been reported as the most predominant genotype in the previous studies. Phylogenetic analysis revealed that G3 rotavirus strains were closely related to the "new variant G3" 5091 strain, which emerged in Japan in 2003-2004. A significant increase in the prevalence of rotavirus G3 found in this study indicates that rotavirus G3 strain is the major cause of infection in five geographical areas of Japan and may distribute globally in the near future.

Three clusters of Saffold viruses circulating in children with diarrhea in Japan.

Saffold virus (SAFV) is a newly discovered human virus in the genus Cardiovirus, family Picornaviridae. The virus was first described from fecal specimens of a child with fever of unknown origin in 2007. A total of 454 fecal specimens were collected from children with diarrhea attended clinics in Japan, 2010-2011, 7 (1.5%) were positive for SAFV. Mixed-infections of SAFV and other enteric viruses (rotavirus, norovirus, and bocavirus) were found in four out of seven cases, while monoinfection by SAFV alone was detected in three cases. In addition to diarrhea, fever and vomiting were observed in three children and mild dehydration in one case. No particular symptoms of cough and rhinorrhea were noted. Analysis of partial VP1 nucleotide sequence of 7 Japanese SAFV strains revealed that 5 SAFV sequences were most closely related with SAFV2 reference strains, but separated into SAFV2-A (3 strains) and SAFV2-B (2 strains). In addition, the other two strains were classified as SAFV3. Our results indicated that SAFVs (SAFV2 and SAFV3) were circulated in children with acute gastroenteritis in Japan during 2010 and 2011 epidemic season.

Detection of human bocaviruses 1 to 4 from nasopharyngeal swab samples collected from patients with respiratory tract infections.

Human bocaviruses (HBoV) 1, 2, 3, and 4 (HBoV1-4) were detected in 132 (15.5%), 5 (0.6%), 3 (0.4%), and 5 (0.6%) of 850 nasopharyngeal swab samples collected from children with respiratory tract infections, respectively. Out of the 145 HBoV1-4-positive samples, 62 (42.8%) were codetected with other respiratory viruses.

Detection of KI polyomavirus and WU polyomavirus DNA by real-time polymerase chain reaction in nasopharyngeal swabs and in normal lung and lung adenocarcinoma tissues.

Polyomaviruses KI (KIPyV) and WU (WUPyV) were detected from 7 (3.0%) and 38 (16.4%) of 232 children with respiratory tract infections by real-time PCR. The rates of infection by KIPyV and WUPyV alone were 3 of 7 (42.9%) and 20 of 38 (52.6%), respectively. In the other samples, various viruses (human respiratory syncytial virus, human metapneumovirus, human rhinovirus, parainfluenza virus 1 and human bocavirus) were detected simultaneously. One case was positive for KIPyV, WUPyV and hMPV. There was no obvious difference in clinical symptoms between KIPyV-positive and WUPyV-positive patients with or without coinfection. KIPyV was detected in one of 30 specimens of lung tissue (3.3%). Neither of the viruses was detected in 30 samples of lung adenocarcinoma tissue.

Detection and genetic characterization of norovirus infections in children with acute gastroenteritis in Japan, 2007-2009.

BACKGROUND: Noroviruses are a major cause of epidemic gastroenteritis in children and adults. The aim of the present study was to investigate the molecular epidemiology of norovirus gastroenteritis in Japan. METHODS: A total of 954 fecal specimens collected from infants and children with acute gastroenteritis from five different regions (Tokyo, Sapporo, Saga, Osaka, and Maizuru) of Japan during 2007-2009 were identified by multiple RT-PCR and semi-nested PCR. RESULTS: Norovirus was detected in a relatively high detection rate (26.6%; 254 of 954). Of the identified NoV, 9.5% (91 of 954) were positive by semi-nested PCR. Norovirus GII (97.3%) was more prevalent than GI (2.7%). Norovirus infections were very common in the patients aged 12-23 months (44.5%; 113 of 254). Winter month seasonality supported norovirus infection in Japan. All 7 GI sequences (100%) detected only in 2007-2008 clustered with Chiba 407 known as GI.4 genotype. Most of the norovirus GII sequences in 2007-2008 belonged to GII.4 (77.9%), followed by GII.14 (11.9%), and GII.3 and GII.6 (5.1% each). In 2008-2009, norovirus sequences were classified into eight distinct genotypes (GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII12, and GII.14). GII.4/2006b variant was responsible for 100% among the detected GII.4 strains in both seasons. Interestingly, GII.6/GII.14 recombinant strains emerged, for the first time in Japanese children, as the second prevalent genotype (11.9%) in 2007-2008 and then dropped rapidly to 2.3% in a year after. In addition, GII.b/GII.3 and GII.4/GII.3 recombinant strains that had been described previously were also found in this study. CONCLUSIONS: This is the first report to demonstrate the co-circulation of the predominant GII.4/2006b variant and the emerging GII.6/GII.14 recombinant strains and supports the importance of norovirus as a causative agent of diarrhea in Japanese children with acute gastroenteritis.